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DNA Lab Helps Return Servicemembers to their Families

By Fred W. Baker IIIAmerican Forces Press Service

WASHINGTON, Sept. 30, 2008  Thumb-tacked to the inside of Jennifer O’Callaghan’s office cubicle is a picture of Marine Capt. William Francis Mullen.

Mitochondrial DNA analysts Christina Miller, right, Jamie Steinitz, center, and Jennifer Kappeller work to prepare bone and tooth samples for DNA extraction. The DNA lab is one of the oldest and largest labs in the world that works with ancient DNA testing, or testing from severely degraded samples. DoD photo by Fred W. Baker III(Click photo for screen-resolution image);high-resolution image available.

On her desk is a red metal bracelet engraved with his name, and the date the fighter pilot went missing.

Mullen presumably was shot down during a combat mission in Laos in 1966. His body was never found, but O’Callaghan hopes to change that.

“Hopefully, one day, we will find him, too,” she said.

O’Callaghan works as a mitochondrial DNA analyst at the Armed Forces DNA Identification Laboratory in Rockville, Md. She is not connected to Mullen in any way other than by the passion that drives her and the others at the lab to painstakingly work through extracting and sequencing the mitochondrial DNA of 800 bone and tooth samples each year provided by the Joint POW/MIA Accounting Command, or JPAC.

DNA testing, once deemed unreliable by the scientific community, has developed to become a key piece of evidence in nearly 85 percent of all missing troop identifications, DoD officials said.

“Once people realized the value of the DNA for forensic examinations and identifications of degraded or unrecognizable remains, it solidified DNA’s role in human identification,” said Army Lt. Col. Louis Finelli, chief deputy medical examiner and director of the DoD DNA registry.

Five teams of technicians, analysts and supervisors work on the cases that are flown in from the JPAC, which is based in Hawaii. The JPAC is one of a handful of DoD agencies charged with recovering and identifying missing servicemembers from past wars. Its Central Identification Lab sends the bone and tooth samples to the DNA lab even as its forensic anthropologists work to identify the remains of recovered servicemembers using other means, such as dental and personnel records, personal items and historical accounts.

The DNA lab has two main missions. One is to provide DNA testing on the remains of current servicemembers who die, and the other is to provide DNA testing on the remains of servicemembers who have been recovered from past wars. In the latter respect, the DNA lab is one of the oldest and largest labs in the world that works with what’s known as “ancient DNA testing,” or testing from severely degraded samples. It also is the world’s leader in working with mitochondrial DNA, officials said.

Nuclear DNA testing is most commonly used now in forensic labs around the world to pinpoint a specific identity. But the drawback is that it can be used mostly only on high-quality samples, and other appropriate DNA references by which to confirm the identity are necessary, such as samples from immediate kin or a blood reference sample.

Mitochondrial DNA, or mtDNA, on the other hand, points only to maternal lineage, but it is easier to extract from severely degraded bone and tooth samples, and reference samples can be from any maternal relative, regardless of the generation. This is especially helpful for identifying servicemembers’ remains from past wars, because some remains are decades old and many immediate family members of the servicemembers have since died.

The development of using mtDNA testing to aid identification of servicemembers has led to a boom in business for the lab. It has shot up from testing only about 200 samples a year in 1995 to having plans to test as many as 1,000 a year by 2010. Also, since the early 1990s, the lab has processed 12,000 DNA reference samples from family members. Their sample results are entered into a database that is referenced against the missing servicemembers’ DNA test results.

Finelli said the mission of the lab speaks to DoD’s efforts to recover its missing servicemembers.

“I think we’re unrivaled in that capacity in the world,” Finelli said.

The sampling process can take months, or sometimes years, depending on the quality of the sample. The scientists there work in the “blind,” not knowing many of the particulars surrounding the cases of those they are testing. Bone and tooth samples are sent to the lab in a small cardboard envelope sealed with evidence tape to protect them from tampering. Inside is a letter with a case and sample number, the conflict and region from which the sample is returned.

JPAC’s recovery teams travel on about 70 missions a year to some of the most remote locations around the world to recover the remains. The DNA lab has received samples from drained rice paddies, termite mounds, glaciers and the sides of mountains, said Jackie Raskin-Burns, a supervisory DNA analyst at AFDIL. She said when remains arrive, the scientists can’t help but wonder who they are and what their story was.

“Every case is different, and every case is exciting,” Raskin-Burns said. She has worked at the lab for 14 years, and said the cases become personal for those who work on them. She keeps track of all cases she works on and matches them with the DoD notifications released after the families are notified.

“I still get goose bumps when a sequence matches a reference,” she said. “I still get a little pit in my stomach when it doesn’t, because you so want to help bring these guys home.”

The samples provided are small -- typically only about 5 grams, weighing less than one-half of an ounce. The surface of each sample is sanded and cleaned to remove external contaminates. Technicians take about half of the sample, about the size of a pea, and grind it into a fine powder. Only about two-tenths of a gram of the powder is used for sampling.

The samples are purified, and everything in the cell is removed except for the pure mtDNA. The target area of the mtDNA is then replicated within the sample to give the scientists a sufficient number of copies to work with. Hundreds of thousands of copies of the targeted mtDNA are made. When finished, the mtDNA is separated and actually is visible on a gel when illuminated by an ultraviolet light.

Once the scientists verify that mtDNA has been obtained and that the sample is clean and accurate, it again is purified and then broken down into its base pair components. Each of the components is fluorescently tagged, allowing the analysts to record the exact order of the components.

The order of these components -- the mitochondrial DNA sequence -- is compared to a reference sequence. Each difference in the sequence is noted, and those differences are compared to a database of other reference sequences.

Mitochondrial DNA is not exclusive to an individual, and some sequences are very common. Therefore, a sequence match does not necessarily prove relationship, because others can have the same sequence. But, if two sequences do not match, it does prove they are not related maternally.

So, if anthropologists recover remains from an airplane crash, they already think they know who was on the plane. When tested, if the mtDNA sequence matches that servicemember’s family reference sample, it is strong evidence that the sample, indeed, came from the person they believed to be on the plane. The test results are combined with other evidence gathered at the crash site and by forensic anthropologists to make an identification.

On the other hand, if the mtDNA sequence from the recovered remains does not match that of a maternal relative of the servicemember thought to be on the plane, the scientists can conclude that the remains are not those they believed them to be.

Once the sequence comparisons are made, a report is sent to the Central Identification Laboratory at the JPAC. If needed, it will return the samples to have them compared to relatives in the family reference sample database. The DNA lab does not make any servicemember identifications. It simply renders a report that indicates whether sample sequences are consistent with reference samples, or that a relationship cannot be determined.

Much of the work is done in test tubes in sterile labs by scientists clad in white lab coats and goggles. Air locks and separate ventilation systems keep stray DNA and other contaminants from ruining the tests. Despite what could be a cold and impersonal scientific environment, many at the lab say they feel personally connected to the mission and are determined to help return the remains to their families.

“If a case doesn’t look like it’s working, I’ll put that much more effort into trying to get something out of it, because I know this is somebody’s relative,” O’Callaghan said. “This is somebody’s brother. This is somebody’s father. And I can be the one who can help bring him home.”

O’Callaghan picked Mullen’s name and profile from the Vietnam War Memorial when she started working at the lab. Her husband is a former Marine who served in combat, and O’Callaghan said that as a family member, she appreciates the lab’s mission.

“I would want somebody to do this for me if something happened to him in Iraq,” she said. “So I am very happy to do this for someone else.”

Raskin-Burns has attended about a dozen presentations of servicemembers’ remains to their families. She said the look on the family members’ faces has kept her at the lab for 14 years.

“When you’re working so hard at one thing for such a long time, to be rewarded by having this person go home to their family, it’s very touching and becomes very personal,” she said.

As a soldier, Finelli said that DoD’s efforts are well spent, and even owed, to servicemembers’ families and to those who have died in combat.

“The parents entrust their sons and daughters to the government every day. It’s only right that we provide an answer back to them should [their sons and daughters] pay the ultimate sacrifice in the line of service to the country,” Finelli said. “Nobody wants to be the lonely soldier out on the battlefield, not embraced by their country, who they died for.”

Finelli said he expects that the lab’s mission will continue to grow as research and technology continue to break new ground in identifying servicemembers’ remains. The lab now leads the way in mtDNA research for the rest of the world, he said, noting that private organizations and other governments have asked for help in many high-profile cases in the past decade. And DoD is investigating expanding the use of DNA testing in the current war on terror, Finelli said.

With the advances in technology and science, Finelli said, he thinks the days of returning servicemembers’ remains to their families decades after they died on the battlefield are over.

“There will never be an unknown soldier out there ever again,” Finelli said. “The government has put their trust and funding into making sure they can tell my parents who I am.”

Carla Paintner, a supervisory DNA analyst at the Armed Forces DNA Identifications Lab in Rockville, Md., holds a test tube of a finely ground bone sample ready to begin the process of extracting the mitochondrial DNA. The lab samples the mitochondrial DNA of 800 bone and tooth samples a year provided by the Joint POW/MIA Accounting Command. DoD photo by Fred W. Baker IIIDownload screen-resolutionDownload high-resolution

The picture and profile of Marine Capt. William Francis Mullen is thumb-tacked to the inside of Jennifer O’Callaghan’s office cubicle at the Armed Forces DNA Identifications Lab in Rockville, Md. O’Callaghan works as a mitochondrial DNA analyst at the lab and hopes that her work will lead to the eventual identification of the missing fighter pilot’s remains. Mullen presumably was shot down during a combat mission in Laos in 1966. DoD photo by Fred W. Baker IIIDownload screen-resolutionDownload high-resolution

Jennifer O’Callaghan, a mitochondrial DNA analyst at the Armed Forces DNA Identifications Lab in Rockville, Md., explains sequencing and how it compares to a reference sample. Mitochondrial DNA points only to maternal lineage, but it is easier to extract from severely degraded bone and tooth samples. DoD photo by Fred W. Baker IIIDownload screen-resolutionDownload high-resolution

Mitochondrial DNA technicians Skip Graf and Allison Fain work in the amplification lab at the Armed Forces DNA Identifications Lab in Rockville, Md. The target area of mitochondrial DNA is replicated within the sample to give the scientists sufficient copies to work with. DoD photo by Fred W. Baker IIIDownload screen-resolutionDownload high-resolution

Mitochondrial DNA analyst Christina Miller sands a bone sample to remove surface contaminants before grinding it into a fine powder and extracting its DNA. DNA testing has developed to become a key piece of evidence in nearly 85 percent of all missing troop identifications. DoD photo by Fred W. Baker IIIDownload screen-resolutionDownload high-resolution

Mitochondrial DNA technician Timothy Herbert sets up samples for sequencing at the Armed Forces DNA Identifications Lab in Rockville, Md. The order of the mitochondrial DNA sequence is compared to a reference sequence. Each difference in the sequence is noted and those differences are compared to a database of other reference sequences. DoD photo by Fred W. Baker IIIDownload screen-resolutionDownload high-resolution

An ultraviolet light shows the amplified mitochondrial DNA on an agarous gel held by technician Chris Johnson. Hundreds of thousands of copies of the target DNA are replicated during the process. DoD photo by Fred W. Baker IIIDownload screen-resolutionDownload high-resolution

Mitochondrial DNA technician Brittany Box readies a sample to amplify its target DNA. The target area is duplicated within the sample to give the scientists sufficient copies to work with. DoD photo by Fred W. Baker IIIDownload screen-resolutionDownload high-resolution

Mitochondrial DNA technician Brittany Box loads a plate of samples into a thermal cycler that will replicate the target DNA. The development of using mitochondrial DNA testing to aid identification of servicemembers has increased in the lab from only about 200 samples a year in 1995 to 800 now. The lab plans to test as many as 1,000 a year by 2010. DoD photo by Fred W. Baker III
Download screen-resolutionDownload high-resolution